human dna methyltransferase 1  (Absolute Biotech Inc)

Journal: International Journal of Molecular Sciences

Article Title: Z-DNA as a Tool for Nuclease-Free DNA Methyltransferase Assay

doi: 10.3390/ijms222111990

Figure Lengend Snippet: Fractions of Z-form of an initially UM or HM CG core after 2-h incubation with various concentrations of M.SssI (1–200 U/mL) or DNMT1 (20–400 U/mL), respectively. The populations were measured in the presence of 50 mM Mg 2+ . FRET efficiency histograms for cores treated with various concentrations of DMT are shown in . ( A ) Fraction of Z-form vs. [M.SssI] (black) overlaid with the effective number of methylated cytosines (blue). ( B ) Fraction of Z-form vs. [DNMT1] (black) overlaid with the effective number of methylated cytosines (blue).

Article Snippet: We purchased both M.SssI (DNA methylase from Spiroplasma sp. strain MQ1) and human DNA methyltransferase 1 (DNMT1) from NEB (New England Biolabs, Inc., Ipswich, MA, USA).

Techniques: Incubation, Methylation

Journal: International Journal of Molecular Sciences

Article Title: Z-DNA as a Tool for Nuclease-Free DNA Methyltransferase Assay

doi: 10.3390/ijms222111990

Figure Lengend Snippet: Natural dietary compounds suppress cytosine methylation in cores efficiently as revealed by reduced Z-DNA formation in the samples treated with those compounds. The concentration of SAM used was 160 μM. FRET efficiency histograms for cores incubated with DMT in the presence of various kinds and concentrations of natural inhibitors are shown in . ( A , B ) DOI of DMT activity by EGCG at various concentrations (( A ) 5, 10, 50, 100, 200, 400, 800 μM for M.SssI and ( B ) 5, 20, 50, 100, 200, 800, 1000 μM for DNMT1). ( C , D ) DOI of DMT activity by curcumin at various concentrations (( C ) 0.5, 1, 5, 10, 800 μM for M.SssI and ( D ) 0.25, 1, 5, 10, 40, 100, 200 μM for DNMT1). ( E , F ) DOI of DMT activity by genistein at various concentrations (( E ) 200, 400, 600, 800 μM for M.SssI and ( F ) 50, 200, 500, 1000, 1800 μM for DNMT1). For EGCG and curcumin, the DOI values for low inhibitor concentrations are presented in insets with the inhibitor concentration in log scale.

Article Snippet: We purchased both M.SssI (DNA methylase from Spiroplasma sp. strain MQ1) and human DNA methyltransferase 1 (DNMT1) from NEB (New England Biolabs, Inc., Ipswich, MA, USA).

Techniques: Methylation, Concentration Assay, Incubation, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Z-DNA as a Tool for Nuclease-Free DNA Methyltransferase Assay

doi: 10.3390/ijms222111990

Figure Lengend Snippet: ( A ) Sample designs and main reactions of this work. DNA tether molecules with a 22-bp CpG repeat (green dotted box) with various degrees of cytosine methylation (bases are color-coded; A: yellow; T: indigo; G: teal; C: red; and mC (methylcytosine): purple) were prepared by hybridizing two dye-labeled complementary oligonucleotides (Cy3 (green ball) and Cy5 (red ball) on each oligonucleotide; FRET dye pairs were separated by 14 base pairs; See , for sequence information) with a specific number of methylcytosines. Hemi-methylated (HM) and unmethylated (UM) molecules were the substrates for, and converted to, full methylated molecules (FM) by DNMT1 and M.SssI, respectively. DNA molecules were treated with DNA methyltransferases (DMT: DNMT1 or M.SssI) in a test tube. DMT enzymes changed cytosines (red) to methylcytosines (purple) by converting SAM to SAH and increased the degree of cytosine methylation in the DNA substrates as shown in the blue box. The reaction mixture was supplemented with one of the natural DMT inhibitors (EGCG, curcumin, or genistein) as shown in the red box. ( B ) Experimental setup and single-molecule detection strategy. Dye-labeled DNA tethers immobilized in the sample chamber are imaged in a TIRF microscope to measure FRET efficiency. The B-Z transition occurring to methylated DNA can be detected via a change in FRET efficiency in Mg 2+ -rich solutions. Representative FRET efficiency histograms for B-DNA (with low [Mg 2+ ]; E FRET ~0.5) and Z-DNA (with high [Mg 2+ ]; E FRET ~0.15) are shown as well. HM, UM, and QM tethers were immobilized on a glass substrate with biotin (blue ball) labeled at the 3′ end of the Cy3-labeled oligonucleotide while TM and FM tethers were immobilized via a biotinylated PCR fragment, which was ligated to the overhang (GATC) from the Cy5 labeled oligonucleotide (CG1 or methylated CG1) of TM or FM.

Article Snippet: We purchased both M.SssI (DNA methylase from Spiroplasma sp. strain MQ1) and human DNA methyltransferase 1 (DNMT1) from NEB (New England Biolabs, Inc., Ipswich, MA, USA).

Techniques: Methylation, Labeling, Sequencing, Microscopy